The biological impact of 1,25-dihydroxyvitamin D3 on cellular proliferation and differentiation and on mature cell function is typical of most steroid-like hormones. Not surprisingly, the mechanism by which the vitamin/hormone functions is also similar. The central role of the vitamin D receptor (VDR) in transducing systemic 1,25-dihydroxyvitamin D3 signals into the target cell nucleus is now well recognized. Despite these insights, it is clear that vitamin D through its receptor exerts highly selective actions on both cells and genes, activities that are dependent upon additional nuclear protein components that function in highly cell- and gene promoter-selective ways. The goal of this project is to further define both general as well as specific features of the vitamin D signaling pathway, features that determine tissue-selective action. The Specific Aims of the project are to: 1) establish the validity and characterize the nature of the VDR modulatory unit (VDR-MU); 2) identify and characterize comodulating amplifiers and/or repressors; and 3) clarify the mechanism by which the inducing actions of vitamin D3 are integrated with other incoming extracellular signals to regulate the expression of human osteocalcin and 24OHase genes. Advanced yeast genetic selection systems will be used to characterize the VDR-MU as well as to identify comodulators of vitamin D3 action. Human homologues will be identified through standard cloning techniques. Both cell transfection and biochemical assays will be utilized to examine the activity of mutant VDRs, cofactor enhancement or repression of these activities, and modulation by unique retinoid receptor ligands. The natural human osteocalcin and 24OHase promoters will be utilized in transiently and stably transfected cells to examine at the promoter level the integration of multiple signaling pathways on vitamin D action. The therapeutic value of 1,25(OH)2D3 in a variety of pathophysiological states has been demonstrated. Moreover, newly synthesized analogues show promise for the treatment of hyperproliferative disorders that are either dermatologic and/or oncologic in nature. Nevertheless, identification of the proteins and mechanism by which selectivity is generated will enhance our understanding of the specific actions of current compounds and enable discovery of new vitamin D analogues and mimetics that exhibit even greater potency, efficacy and tissue selectivity.